Fructan Biosynthetic Enzymes

ABSTRACT

This invention relates to isolated nucleic acid fragments encoding fructosyltransferases. More specifically, this invention relates to polynucleotides encoding 1-FFTs, 6-SFTs, or 1-SSTs. The invention also relates to the construction of a recombinant DNA constructs encoding all or a portion of the fructosyltransferases, in sense or antisense orientation, wherein expression of the recombinant DNA construct results in production of altered levels of the fructosyltransferases in a transformed host cell.

This application is a Continuation of U.S. application Ser. No.10/916,247, filed Aug. 11, 2004, now U.S. Pat. No. 7,253,335, which is aDivisional of U.S. application Ser. No. 10/003,392, filed Oct. 30, 2001,now U.S. Pat. No. 6,791,015, which claims the benefit of U.S.Provisional Application No. 60/244,273, filed Oct. 30, 2000, and U.S.Provisional Application No. 60/269,543, filed Feb. 16, 2001. The entirecontents of these applications are herein incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingfructosyltransferases in plants and seeds.

BACKGROUND OF THE INVENTION

Fructans are linear or branched polymers of repeating fructose residueswith usually one terminal glucose unit. The number of residues containedin an individual polymer, also known as the degree of polymerization(DP), varies greatly depending on the source from which the polymer isisolated. Several bacteria can produce fructans with a DP 5000 orgreater, while low DP fructans (DP 3 to 200) are found in over 40,000plant species.

Based on their structure, several types of fructans can be identified inhigher plants. The most characterized plant fructan is inulin. Inulincontains linear β(2-1)-linked fructosyl residues and commonly occurs inthe Asterales such as Jerusalem artichoke (Helianthus tuberosus),sunflower (Helianthus sp.), Belgian endive (Cichorium intybus) andartichoke (Cynara scolymus). Inulin synthesis is initiated bysucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) whichcatalyses the conversion of sucrose into isokestose (also named1-kestose) and glucose. Additional fructosyl units are added ontoisokestose, by the action of a fructan:fructan 1-fructosyltransferase(1-FFT, EC 2.4.1.100) resulting in a β(2-1)-linked fructose oligomer.

A second type of fructan is called levan and consists of linear β(2-6)linked fructosyl residues. Grasses such as Dactylis glomerata and Phleumpratense contain levans with a DP up to 200. Levans are synthesized by asucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10) that usessucrose as a fructosyl donor and acceptor to produce 6-kestose.Polymerization of 6-kestose is believed to be catalyzed by 6-SFT aswell, using sucrose as the fructosyl donor.

A third type of fructan, graminan (also called mixed-levan), is found inmany Poales such as barley and wheat. These plants use an SST to produceiso-kestose from sucrose, and 6-SFT to further polymerize isokestose,resulting in a fructan containing both the β(2-1) and the β(2-6) linkedfructosyl residues.

The fourth type of fructan is often referred to as the neo-kestoseseries of fructans. The neo-kestose series have fructosyl residues onthe carbon 1 and 6 of glucose producing a polymer with fructosylresidues on either end of the sucrose molecule. The inulin-neoseriesfound in Liliales such as onion (Allium cepa), leek (Allium porrum), andasparagus (Asparagus officinales) contain mainly a β(2-1)-linkedfructose polymer linked to carbon 1 and 6 of glucose, while thelevan-neoseries contain mainly a β(2-6)-linked fructose polymer linkedto carbon 1 and 6 of glucose. Neoseries fructans are believed to besynthesized by the concerted action of 1-SST (producing isokestose) and6G-FFT, a specific fructan:fructan 6G-fructosyltransferase thatpolymerizes fructosyl units onto carbon 6 of glucose.

Industrial applications of fructans are very diverse and range frommedical, food, and feed applications, as well as the use of fructans asa raw material for the production of industrial polymers andhigh-fructose syrup. Regardless of size, fructose polymers are notmetabolized by humans and animals. Fructans can enhance animal healthand performance by being selectively fermented by beneficial organismssuch as Bifidibacterium in the large intestine of animals, at theexpense of pathogenic organisms such as E. coli and Salmonella, leadingto altered fatty acid profiles, increased nutrient absorption, anddecreased levels of blood cholesterol. Also, fructans have a sweet tasteand are increasingly used as low-calorie sweeteners and as functionalfood ingredients.

Accordingly, there is a great deal of interest in understanding fructanbiosynthetic pathways. With the isolation of nucleic acid fragmentsencoding various enzymes involved in the pathway, it may be possible toengineer transgenic plants to produce desired levels of different typesof useful and novel fructans.

SUMMARY OF THE INVENTION

The present invention concerns an isolated polynucleotide comprising:(a) a first nucleotide sequence encoding a first polypeptide comprisingat least 58 amino acids, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:12 have at least90% or 95% identity based on the Clustal alignment method, (b) a secondnucleotide sequence encoding a second polypeptide comprising at least140 amino acids, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:6 have at least 90%or 95% identity based on the Clustal alignment method, (c) a thirdnucleotide sequence encoding a third polypeptide comprising at least 471amino acids, wherein the amino acid sequence of the third polypeptideand the amino acid sequence of SEQ ID NO:10 have at least 95% identitybased on the Clustal alignment method, (d) a fourth nucleotide sequenceencoding a fourth polypeptide comprising at least 495 amino acids,wherein the amino acid sequence of the fourth polypeptide and the aminoacid sequence of SEQ ID NO:8 have at least 95% identity based on theClustal alignment method, (e) a fifth nucleotide sequence encoding afifth polypeptide comprising at least 600 amino acids, wherein the aminoacid sequence of the fifth polypeptide and the amino acid sequence ofSEQ ID NO:2 have at least 85%, 90%, or 95% identity based on the Clustalalignment method, (f) a sixth nucleotide sequence encoding a sixthpolypeptide comprising at least 600 amino acids, wherein the amino acidsequence of the sixth polypeptide and the amino acid sequence of SEQ IDNO:4 or SEQ ID NO:14 have at least 90% or 95% identity based on theClustal alignment method, (g) a seventh nucleotide sequence encoding aseventh polypeptide comprising at least 630 amino acids, wherein theamino acid sequence of the seventh polypeptide and the amino acidsequence of SEQ ID NO:16 have at least 97% identity based on the Clustalalignment method, or (h) the complement of the first, second, third,fourth, fifth, sixth, or seventh nucleotide sequence, wherein thecomplement and the first, second, third, fourth, fifth, sixth, orseventh nucleotide sequence contain the same number of nucleotides andare 100% complementary.

In a second embodiment, the first polypeptide preferably comprises theamino acid sequence of SEQ ID NO:12, the second polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:6, the third polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:10, the fourthpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:8,the fifth polypeptide preferably comprises the amino acid sequence ofSEQ ID NO:2, the sixth polypeptide preferably comprises the amino acidsequence of SEQ ID NO:4 or SEQ ID NO:14, and the seventh polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:16.

In a third embodiment, the first nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:11, the second nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:5,the third nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:9, the fourth nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:7, the fifth nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:1,the sixth nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:3 or SEQ ID NO:13, and the seventh nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:15.

In a fourth embodiment, the first, second, third, fourth, fifth, sixth,and seventh polypeptides preferably are fructosyltranferases.

In a fifth embodiment, the first, third and fourth polypeptidespreferably are 6-SFT, the second and fifth polypeptides preferably are1-FFT, the sixth polypeptide preferably is 1-FFT or 1-SST, and theseventh polypeptide preferably is 1-SST.

In a sixth embodiment, this invention relates to a vector comprising thepolynucleotide of the present invention, or to a recombinant DNAconstruct comprising the polynucleotide of the present inventionoperably linked to at least one regulatory sequence. The inventionincludes a cell, a plant, or a seed comprising the recombinant DNAconstruct of the present invention. The cell may be a eukaryotic cellsuch as a plant cell, or a prokaryotic cell such as a bacterial cell.

In a seventh embodiment, the invention relates to a virus, preferably abaculovirus, comprising an isolated polynucleotide of the presentinvention or a recombinant DNA construct of the present invention.

In an eighth embodiment, the invention relates to a method oftransforming a cell by introducing into the cell a nucleic acidcomprising a polynucleotide of the present invention. The invention alsoconcerns a method for producing a transgenic plant comprisingtransforming a plant cell with any of the isolated polynucleotides ofthe present invention and regenerating a plant from the transformedplant cell, the transgenic plant produced by this method, and the seedobtained from this transgenic plant.

In a ninth embodiment, the present invention relates to (a) a method forproducing a polynucleotide fragment comprising selecting a nucleotidesequence comprised by any of the polynucleotides of the presentinvention, wherein the selected nucleotide sequence contains at least30, 40, or 60 nucleotides, and synthesizing a polynucleotide fragmentcontaining the selected nucleotide sequence, and (b) the polynucleotidefragment produced by this method.

In a tenth embodiment, the present invention relates to an isolatedpolynucleotide fragment comprising a nucleotide sequence comprised byany of the polynucleotides of the present invention, wherein thenucleotide sequence contains at least 30, 40, or 60 nucleotides, and acell, a plant, and a seed comprising the isolated polynucleotide.

In an eleventh embodiment, the present invention concerns an isolatedpolypeptide comprising: (a) a first amino acid sequence comprising atleast 58 amino acids, wherein the first amino acid sequence and theamino acid sequence of SEQ ID NO:12 have at least 90% or 95% identitybased on the Clustal alignment method, (b) a second amino acid sequencecomprising at least 140 amino acids, wherein the second amino acidsequence and the amino acid sequence of SEQ ID NO:6 have at least 90% or95% identity based on the Clustal alignment method, (c) a third aminoacid sequence encoding comprising at least 471 amino acids, wherein thethird amino acid sequence and the amino acid sequence of SEQ ID NO:10have at least 95% identity based on the Clustal alignment method, (d) afourth amino acid sequence comprising at least 495 amino acids, whereinthe fourth amino acid sequence and the amino acid sequence of SEQ IDNO:8 have at least 95% identity based on the Clustal alignment method,(e) a fifth amino acid sequence comprising at least 600 amino acids,wherein the fifth amino acid sequence and the amino acid sequence of SEQID NO:2 have at least 85%, 90%, or 95% identity based on the Clustalalignment method, (f) a sixth amino acid sequence comprising at least600 amino acids, wherein the sixth amino acid sequence and the aminoacid sequence of SEQ ID NO:4 or SEQ ID NO:14 have at least 90% or 95%identity based on the Clustal alignment method, or (g) a seventh aminoacid sequence comprising at least 630 amino acids, wherein the seventhamino acid sequence and the amino acid sequence of SEQ ID NO:16 have atleast 97% identity based on the Clustal alignment method. The firstamino acid sequence preferably comprises the amino acid sequence of SEQID NO:12, the second amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:6, the third amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:10, the fourth amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:8,the fifth amino acid sequence preferably comprises the amino acidsequence of SEQ ID NO:2, the sixth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:14, andthe seventh amino acid sequence preferably comprises the amino acidsequence of SEQ ID NO:16. The polypeptide preferably is afructosyltranferase. The first, third and fourth amino acid sequencespreferably are 6-SFT, the second and fifth amino acid sequencespreferably are 1-FFT, the sixth amino acid sequence preferably is 1-FFTor 1-SST, and the seventh amino acid sequence preferably is 1-SST.

In a twelfth embodiment, the invention concerns a method for isolating apolypeptide encoded by the polynucleotide of the present inventioncomprising isolating the polypeptide from a cell containing arecombinant DNA construct comprising the polynucleotide operably linkedto a regulatory sequence.

In a thirteenth embodiment, the invention relates to a method ofselecting an isolated polynucleotide that affects the level ofexpression of a fructan biosynthetic enzyme (fructosyltransferase)polypeptide or enzyme activity in a host cell, preferably a plant cell,the method comprising the steps of: (a) constructing an isolatedpolynucleotide of the present invention or an isolated recombinant DNAconstruct of the present invention; (b) introducing the isolatedpolynucleotide or the isolated recombinant DNA construct into a hostcell; (c) measuring the level of the fructan biosynthetic enzyme(fructosyltransferase) polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide or the isolated recombinant DNAconstruct; and (d) comparing the level of the fructan biosyntheticenzyme (fructosyltransferase) polypeptide or enzyme activity in the hostcell containing the isolated polynucleotide or the isolated recombinantDNA construct with the level of the fructan biosynthetic enzyme(fructosyltransferase) polypeptide or enzyme activity in the host cellthat does not contain the isolated polynucleotide or the isolatedrecombinant DNA construct.

In a fourteenth embodiment, the invention concerns a method of obtaininga nucleic acid fragment encoding a substantial portion of a fructanbiosynthetic enzyme (fructosyltransferase) polypeptide, preferably aplant fructan biosynthetic enzyme (fructosyltransferase) polypeptide,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 30 (preferably atleast one of 40, most preferably at least one of 60) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15 and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a substantial portion of a fructan biosynthetic enzyme(fructosyltransferase) polypeptide amino acid sequence.

In a fifteenth embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a fructan biosynthetic enzyme(fructosyltransferase) polypeptide comprising the steps of: probing acDNA or genomic library with an isolated polynucleotide of the presentinvention; identifying a DNA clone that hybridizes with an isolatedpolynucleotide of the present invention; isolating the identified DNAclone; and sequencing the cDNA or genomic fragment that comprises theisolated DNA clone.

In a sixteenth embodiment, this invention concerns a composition, suchas a hybridization mixture, comprising an isolated polynucleotide of thepresent invention.

In a seventeenth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the recombinant DNA construct of the present invention oran expression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the fructanbiosynthetic enzyme (fructosyltransferase) polypeptide in an amountsufficient to complement a null mutant to provide a positive selectionmeans.

In an eighteenth embodiment, this invention relates to a method ofaltering the level of expression of a fructan biosynthetic enzyme(fructosyltransferase) polypeptide in a host cell comprising: (a)transforming a host cell with a recombinant DNA construct of the presentinvention; and (b) growing the transformed host cell under conditionsthat are suitable for expression of the recombinant DNA constructwherein expression of the recombinant DNA construct results inproduction of altered levels of the fructan biosynthetic enzyme(fructosyltransferase) polypeptide in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A-1C show an alignment of the 1-FFT amino acid sequences encodedby the African daisy clone dms2c.pk006.p1 (SEQ ID NO:2), the guayuleclone epb3c.pk007.j9 (SEQ ID NO:4), and the sunflower clonehss1c.pk004.i5 (SEQ ID NO:6), with the Helianthus tuberosus 1-FFT (NCBIGeneral Identifier No. 3367690; SEQ ID NO:17). Amino acids conservedamong all sequences are indicated with an asterisk (*) above thealignment. The program uses dashes to maximize the alignment. FIG. 1Ashows amino acids 1 through 240, FIG. 1B shows amino acids 241 through480, and FIG. 1C shows amino acids 481 through 624.

FIGS. 2A-2C shows an alignment of the 6-SFT amino acid sequences encodedby by the wheat clone wdk1c.pk014.c11 (SEQ ID NO:8), wheat clonewdk2c.pk017.f14 (SEQ ID NO:10), wheat clone wr1.pk0085.h8 (SEQ IDNO:12), and wheat clone wdk2c.pk017.f14:cgs (SEQ ID NO:20), with theHordeum vulgare sequence (NCBI General Identifier No. 7435467; SEQ IDNO:21). Amino acids conserved among all sequences are indicated with anasterisk (*) above the alignment. The program uses dashes to maximizethe alignment. FIG. 2A shows amino acids 1 through 240, FIG. 2B showsamino acids 241 through 480, and FIG. 2C shows amino acids 481 through625.

FIGS. 3A-3B show an alignment of the 1-SST amino acid sequences encodedby the guayule clone epb3c.pk007.n11 (SEQ ID NO:14) and the sunflowerclone hhs1c.pk004.e5 (SEQ ID NO:16), with the Helianthus tuberosus 1-SST(NCBI General Identifier No. 3367711; SEQ ID NO:18). Amino acidsconserved among all sequences are indicated with an asterisk (*) abovethe alignment. The program uses dashes to maximize the alignment. FIG.3A shows amino acids 1 through 350, and FIG. 3B shows amino acids 351through 637.

Table 1 lists the polypeptides that are described herein (including theplant source from where they are derived), the designation of the cDNAclones that comprise the nucleic acid fragments encoding polypeptidesrepresenting all or a substantial portion of these polypeptides, and thecorresponding identifier (SEQ ID NO:) as used in the attached SequenceListing. The table also includes the art sequences used in the figures,the polypeptide, source, and General Identifier No. (GI No.). Thesequence descriptions and Sequence Listing attached hereto comply withthe rules governing nucleotide and/or amino acid sequence disclosures inpatent applications as set forth in 37 C.F.R. §1.821-1.825.

TABLE 1 Fructosyltransferases SEQ ID NO: (Amino Protein CloneDesignation (Nucleotide) Acid) African Daisy 1-FFT dms2c.pk006.p1 1 2Guayule 1-FFT epb3c.pk007.j9 3 4 Sunflower 1-FFT hss1c.pk004.i5:fis 5 6Wheat 6-SFT wdk1c.pk014.c11 7 8 Wheat 6-SFT wdk2c.pk017.f14 9 10 Wheat6-SFT wr1.pk0085.h8 11 12 Guayule 1-SST epb3c.pk007.n11 13 14 Sunflower1-SST hhs1c.pk004.e5 15 16 H. tuberosus 1-FFT GI No. 3367690 17 H.tuberosus 1-SST GI No. 3367711 18 Wheat 6-SFT wdk2c.pk017.f14:cgs 19 20Hordeum vulgare 6-SFT GI No. 7435467 21

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNOs:1, 3, 5, 13, 15, or 19 or the complement of such sequences.

The term “isolated” refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not alter the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not change the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 13,or 15 and the complement of such nucleotide sequences may be used toaffect the expression and/or function of a fructosyltransferase selectedfrom 1-FFT, 6-SFT and 1-SST in a host cell. A method of using anisolated polynucleotide to affect the level of expression of apolypeptide in a host cell (eukaryotic, such as plant or yeast,prokaryotic such as bacterial) may comprise the steps of: constructingan isolated polynucleotide of the present invention or an isolatedchimeric gene of the present invention; introducing the isolatedpolynucleotide or the isolated chimeric gene into a host cell; measuringthe level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IR Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are at least about 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are at least about 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. The amino acidsequences may be 96% identical, 97% identical, 98% identical, 99%identical, or any integer thereof. Suitable nucleic acid fragments notonly have the above identities but typically encode a polypeptide havingat least 50 amino acids, preferably at least 100 amino acids, morepreferably at least 150 amino acids, still more preferably at least 200amino acids, and most preferably at least 250 amino acids. Sequencealignments and percent identity calculations were performed using theMegalign program of the LASERGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences wasperformed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST alogarithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that because in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by effecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns isolated polynucleotides comprising anucleotide sequence encoding a polypeptide having fructosyltransferaseactivity wherein (a) the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:2, 4, or 6 have at least 90% sequenceidentity; (b) the amino acid sequence of the polypeptide and the aminoacid sequence of SEQ ID NO:14 or 16 have at least 97% identity. It ispreferred that the identity in (a) be at least 95%. The presentinvention also relates to isolated polynucleotides comprising thecomplement of the nucleotide sequence, wherein the complement and thenucleotide sequence contain the same number of nucleotides and are 100%complementary. More specifically, the present invention concernsisolated polynucleotides encoding 1-FFT polypeptides having the sequenceof SEQ ID NO:2, 4, or 6, or 1-SST polypeptides having the sequence ofSEQ ID NO:14 or 16.

Nucleic acid fragments encoding at least a portion of severalfructosyltransferases have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. The nucleic acid fragments of the instant inventionmay be used to isolate cDNAs and genes encoding homologous proteins fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other 1-FFTs, 6-SFTs, or 1-SSTs, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 30 (preferably at least 40, most preferably at least 60)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 13, or 15 and the complementof such nucleotide sequences may be used in such methods to obtain anucleic acid fragment encoding a substantial portion of an amino acidsequence of a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the fructan profile in thosecells. Nucleic acid fragments encoding the fructan biosynthetic enzymes(fructosyltransferases) disclosed herein may be used to generatetrangenic plants that produce particular fructans. In particular, theability to produce fructans of the desired size in large amounts incrops of agronomic importance, such as corn or soybean, will reducefructan production costs.

U.S. Pat. No. 5,840,361 teaches the health benefits of baby foodcompositions comprising fructan-containing vegetables. These benefitsare based on the role of fructan-containing foods in stimulating theproduction of beneficial intestinal microbes, such as theBifidobacterium species. Bifidobacteria are thought to promote health bytheir fermentation of sugars in the colon. This activity inhibits thedevelopment of putrefactive bacteria and provides resistance toinfective gastroenteritis (Langhendries et al. (1995) J PedGastroenterol Nutr 21:177-181; Jason et al. (1984) Pediatrics74(Suppl):702-727; Howie et al. (1990) Br Med J 300:11-16). Stimulatingcolonic bifidobacteria may also result in the enhancement of immunefunctions, the improvement of digestion and absorption of essentialnutrients, and the synthesis of vitamins (Gibson et al. (1995) J Nutr125:1401-1412). One approach to increasing the colonic bifidobacteria inhumans is termed prebiotics. Prebiotics involves feeding of anondigestable food ingredient, such as fructooligosaccharides, thatbeneficially affects the microflora by selectively stimulating thegrowth and/or activity of beneficial bacteria. Oral administration tohumans of fructans such as oligofructose and inulin have been shown toincrease the number of bifidobacteria in stools (Gibson et al. (1995)Gastroenterol 108:975-982). Consequently, fructans have been recommendedas dietary supplements to adult humans (Modler et al. (1990) Can InstFood Sci Technol J 23:29-41). The enzymes 1-SST and 1-FFT are involvedin the biosynthesis of inulin and other fructans. The overexpression of1-SST and 1-FFT in crop species such as corn, wheat, rice and soybeanshould facilitate the production of fructan-containing material withbeneficial prebiotic properties.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by amplification of DNA orRNA, Southern analysis of DNA, Northern analysis of mRNA expression,Western analysis of protein expression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate theirsecretion from the cell. It is thus envisioned that the recombinant DNAfragments described above may be further supplemented by directing thecoding sequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns afructosyltransferase polypeptide, most preferably a 1-FFT polypeptidehaving an amino acid sequence that is at least 90% identical, based onthe Clustal method of alignment, to a polypeptide selected from thegroup consisting of SEQ ID NOs:2, 4, and 6. The preferredfructosyltransferase polypeptide may be a 1-SST polypeptide having anamino acid sequence that is at least 97% identical, based on the Clustalmethod of alignment, to a polypeptide selected from the group consistingof SEQ ID NOs:14 and 16.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded fructosyltransferase (1-FFT, 6-SFT, or 1-SST). An example ofa vector for high level expression of the instant polypeptides in abacterial host is provided (Example 8).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

Nucleic acid probes derived from the instant nucleic acid sequences maybe used in direct fluorescence in situ hybridization (FISH) mapping(Trask (1991) Trends Genet. 7:149-154). Although current methods of FISHmapping favor use of large clones (several to several hundred KB; seeLaan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity mayallow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptides. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptides can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various African daisy(Dimorphotheca sinuata), guayule (Parthenium argentatum Grey), sunflower(Helianthus sp.), and wheat (Triticum aestivum) tissues were prepared.The general characteristics of the libraries are indicated below.

TABLE 2 cDNA Libraries from African Daisy, Guayule, Sunflower, and WheatLibrary Tissue Clone dms2c African Daisy Developing Seed dms2c.pk006.p1epb3c Guayule Stem Bark epb3c.pk007.j9 epb3c.pk007.n11 hhs1c SunflowerHead Tissue Infected hhs1c.pk004.e5 With Sclerotinia hss1cSclerotinia-Infected Sunflower Plant hss1c.pk004.i5 wdk1c WheatDeveloping Kernel, 3 Days wdk1c.pk014.c11 After Anthesis wdk2c WheatDeveloping Kernel, 7 Days wdk2c.pk017.f14 After Anthesiswdk2c.pk017.f14:cgs wr1 Wheat Root From 7 Day Old wr1.pk0085.h8 Seedling

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

In some of the clones the cDNA fragment corresponds to a portion of the3′-terminus of the gene and does not cover the entire open readingframe. In order to obtain the upstream information one of two differentprotocols are used. The first of these methods results in the productionof a fragment of DNA containing a portion of the desired gene sequencewhile the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

cDNA clones encoding fructosyltransferases (1-FFT, 6-SFT, or 1-SST) wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST alogarithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health) searches for similarityto sequences contained in the BLAST “nr” database (comprising allnon-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The cDNA sequences obtained in Example 1 were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding 1-FFT

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to 1-FFTfrom Helianthus tuberosus (NCBI General Identifier No. 3367690). Shownin Table 3 are the BLAST results for individual ESTs (“EST”), or for thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”) and encoding the entire protein (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to1-FFT BLAST pLog Score Clone Status NCBI GI No. 3367690dms2c.pk006.p1:fis CGS >180.00 epb3c.pk007.j9:fis CGS >180.00hss1c.pk004.i5 EST 83.70

The nucleotide sequence of the entire cDNA insert in clonedms2c.pk006.p1 is shown in SEQ ID NO:1. The amino acid sequence deducedfrom nucleotides 33 through 1856 of SEQ ID NO:1 is shown in SEQ ID NO:2.The nucleotide sequence of the entire cDNA insert in cloneepb3c.pk007.j9 is shown in SEQ ID NO:3. The amino acid sequence deducedfrom nucleotides 63 through 1889 of SEQ ID NO:3 is shown in SEQ ID NO:4.The nucleotide sequence of a portion of the cDNA insert in clonehss1c.pk004.15 is shown in SEQ ID NO:5. The amino acid sequence deducedfrom nucleotides 1 through 413 of SEQ ID NO:5 is shown in SEQ ID NO:6.

FIGS. 1A-1C present an alignment of the amino acid sequences set forthin SEQ ID NOs:2, 4, and 6 with the Helianthus tuberosus 1-FFT sequence(NCBI General Identifier No. 3367690; SEQ ID NO:17). The alignmentindicates with an asterisk (*) above the alignment the amino acidsconserved among all the sequences. FIG. 1A shows amino acids 1 through240, FIG. 1B shows amino acids 241 through 480, and FIG. 1C shows aminoacids 481 through 624.

According to van der Meer et al. ((1998) Plant J. 15:489-500) theHelianthus tuberosus amino acid sequence has a signal sequencecorresponding to amino acids 1 through 80. It can be clearly seen fromthe alignment that the polypeptides having SEQ ID NO:2 and SEQ ID NO:4contain the conserved domains highlighted by Cha et al. ((2001) J.Biotech. 91:49-61) and the conserved Asp and Glu suggested by Saito etal. ((1997) Biosci. Biotech. Biochem. 61:2076-2079) as playing a role asnucleophile and proton donor in the catalytic mechanism offructosyltransferases. In 1-FFTs the “FRDP-F motif” is included withinthe conserved motifPhe-His-Phe-Gln-Pro-Ala-Lys-Asn-Phe-Ile-Asp-Pro-Xaa-Gly. The “ECPD-Rmotif” is included within the conserved domain His-Ser-Val-Pro-Asn-Thr-Asp-Met-Trp-Glu-Cys-Val-Asp-Phe-Tyr-Pro-Val-Ser-Leu-Thr-Asn-Asp-Ser-Ala-Leu-Asp.The putative active Asp is included in the first domain and is locatedat position 95 of both, SEQ ID NO:2 and SEQ ID NO:4. The conserved Gluis found in the second domain, at position 277 of both, SEQ ID NO:2 andSEQ ID NO:4.

The data in Table 4 presents the percent identity of the amino acidsequences set forth in SEQ ID NOs:2, 4, and 6 with the Helianthustuberosus sequence (NCBI General Identifier No. 3367690; SEQ ID NO:17).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to1-FFT Percent Identity to Clone SEQ ID NO. 3367690 dms2c.pk006.p1:fis 279.5 epb3c.pk007.j9:fis 4 84.9 hss1c.pk004.i5 6 86.5

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode entire African daisy and Guayule 1-FFT and a substantial portionof a sunflower 1-FFT. These sequences represent the first African daisy,guayule, and sunflower sequences encoding 1-FFT known to Applicant.

Example 4 Characterization of cDNA Clones Encoding 6-SFT

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to 6-SFTfrom Hordeum vulgare (NCBI General Identifier No. 7435467). Shown inTable 5 are the BLAST results for the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous to6-SFT BLAST pLog Score Clone Status 7435467 wdk1c.pk014.c11 FIS >180.00wdk2c.pk017.f14 FIS >180.00 wr1.pk0085.h8 FIS 21.30

The nucleotide sequence of the entire cDNA insert in clonewdk1c.pk014.c11 is shown in SEQ ID NO:7. The amino acid sequence deducedfrom nucleotides 3 through 1487 of SEQ ID NO:7 is shown in SEQ ID NO:8.The nucleotide sequence of the entire cDNA insert in clonewdk2c.pk017.f14 is shown in SEQ ID NO:9. The amino acid sequence deducedfrom nucleotides 1 through 1413 of SEQ ID NO:9 is shown in SEQ ID NO:10.The nucleotide sequence of the entire cDNA insert in clone wr1.pk0085.h8is shown in SEQ ID NO:11. The amino acid sequence deduced fromnucleotides 1 through 174 of SEQ ID NO:11 is shown in SEQ ID NO:12.

The nucleotide sequence encoding the N-terminus for the polypeptideencoded by the cDNA insert in clone wdk2c.pk017.f14 was obtained. TheBLASTP search using the amino acid sequence from clones wdk2c.pk017.f14revealed similarity of the 6-SFT polypeptide from Hordeum vulgare (NCBIGeneral Identifier No. 7435467). Shown in Table 6 are the BLAST resultsfor the amino acid sequence of the entire protein encoded by theindicated clone (CGS):

TABLE 6 BLAST Results for Sequences Encoding Polypeptides Homologous to6-SFT BLAST pLog Score Clone Status 7435467 wdk2c.pk017.f14:cgs CGS>180.00

A contig of the nucleotide sequence of the entire cDNA insert in clonewdk2c.pk017.f14 and 5′PCR is shown in SEQ ID NO:19. The amino acidsequence deduced from nucleotides 3 through 1916 of SEQ ID NO:19 isshown in SEQ ID NO:20.

FIGS. 2A-2C present an alignment of the amino acid sequences set forthin SEQ ID NOs:8, 10, 12, and 20 with the Hordeum vulgare sequence (NCBIGeneral Identifier No. 7435467; SEQ ID NO:21). The alignment indicateswith an asterisk (*) above the alignment the amino acids conserved amongall the sequences. Dashes are used by the program to maximize thealignment. FIG. 2A shows amino acids 1 through 240, FIG. 2B shows aminoacids 241 through 480, and FIG. 2C shows amino acids 481 through 625. Itcan be clearly seen from the alignment that the polypeptide having SEQID NO:20 contains both of the conserved domains mentioned in Example 3and that SEQ ID NO:8, 10, and 12 only contain the second motif. In6-SFTs the “FRDP-F motif” is contained within the conserved domainGln-Thr-Ala-Lys-Asn-Tyr-Met-Ser-Asp-Pro-Asn-Gly-Leu-Met-Tyr whichincludes the “active Asp” at position 77 of SEQ ID NO:20. In 6-SFTs the“ECPD-R motif” is included within the domainArg-Thr-Gly-Glu-Trp-Glu-Cys-Ile-Asp-Phe-Tyr-Pro-Val-Gly. The “activeGlu” is found at position 158 of SEQ ID NO:8, at position 134 of SEQ IDNO:10, and at position 263 of SEQ ID NO:20.

The data in Table 7 presents the percent identity of the amino acidsequences set forth in SEQ ID NOs:8, 10, 12, and 20 with the Hordeumvulgare sequence (NCBI General Identifier No. 7435467; SEQ ID NO:21).

TABLE 7 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to6-SFT Percent Identity to Clone SEQ ID NO. 7435467 wdk1c.pk014.c11 894.9 wdk2c.pk017.f14 10 94.7 wr1.pk0085.h8 12 84.5 wdk2c.pk017.f14:cgs20 88.7

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode an entire wheat 6-SFTs and substantial portions of a three wheat6-SFTs. These sequences represent the first wheat sequences encoding6-SFT known to Applicant.

Example 5 Characterization of cDNA Clones Encoding 1-SST

The BLASTX search using the EST sequences from clones listed in Table 8revealed similarity of the polypeptides encoded by the cDNAs to 1-SSTfrom Helianthus tuberosus (NCBI General Identifier No. 3367711). Shownin Table 7 are the BLAST results for the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”) encoding an entireprotein (“CGS”):

TABLE 8 BLAST Results for Sequences Encoding Polypeptides Homologous to1-SST BLAST pLog Score Clone Status NCBI GI No. 3367711 epb3c.pk007.n11CGS >180.00 hhs1c.pk004.e5 CGS >180.00

The nucleotide sequence of the entire cDNA insert in cloneepb3c.pk007.n11 is shown in SEQ ID NO:13. The amino acid sequencededuced from nucleotides 42 through 1946 of SEQ ID NO:13 is shown in SEQID NO:14. The nucleotide sequence of the entire cDNA insert in clonehhs1c.pk004.e5 is shown in SEQ ID NO:15. The amino acid sequence deducedfrom nucleotides 59 through 1890 of SEQ ID NO:15 is shown in SEQ IDNO:16.

FIGS. 3A-3B present an alignment of the amino acid sequences set forthin SEQ ID NOs:14 and 16 with the Helianthus tuberosus 1-SST sequence(NCBI General Identifier No. 3367711; SEQ ID NO:18). The alignmentindicates with an asterisk (*) above the alignment the amino acidsconserved among all the sequences. According to van der Meer et al.((1998) Plant J. 15:489-500) the mature Helianthus peptide consists ofamino acids 100 through 630. FIG. 3A shows amino acids 1 through 350,and FIG. 3B shows amino acids 351 through 637.

It can be clearly seen from the alignment that the polypeptides havingSEQ ID NO:14 and SEQ ID NO:16 contain conserved domains which includethe motifs mentioned in Example 3. In 1-SSTs the “FRDP-F motif” iscontained within the conserved domainTyr-His-Phe-Gln-Pro-Asp-Lys-Xaa-Ile-Ser-Asp-Pro-Asp-Gly-Pro-Met-Tys-Hiswhich includes the “active Asp” at position 115 of SEQ ID NO:14 and atposition 108 of SEQ ID NO:16. In 1-SSTs the “ECPD-R motif” is includedwithin the domainGlu-Glu-Val-Leu-His-Ala-Val-Pro-His-Thr-Gly-Met-Trp-Asp-Cys-Val-Asp-Leu-tyr-Pro.The “active Glu” is found at position 293 of SEQ ID NO:8 and at position286 of SEQ ID NO:20.

The data in Table 9 presents the percent identity of the amino acidsequences set forth in SEQ ID NOs:14 and 16 with the Helianthustuberosus sequence (NCBI General Identifier No. 3367711; SEQ ID NO:18).

TABLE 9 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequencesof cDNA Clones Encoding Polypeptides Homologous to1-SST Percent Identity to Clone SEQ ID NO. 3367711 epb3c.pk007.n11 1489.2 hhs1c.pk004.e5 16 96.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode entire 1-SSTs. These sequences represent the first sunflower andguayule sequences encoding 1-SST known to Applicant.

Example 6 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialophos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialophos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialophos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).Assays for fructosyltransferase activity may be conducted under wellknown experimental conditions which permit optimal enzymatic activity.For example, assays for 1-FFT and 1-SST are presented by van der Meer etal. (1998) Plant J. 15:489-500. Assays for 6-SFT are presented bySprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92:11652-11656.

Example 7 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the Rsubunit of the seedstorage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al.(1986) J. Biol. Chem. 261:9228-9238) can be used for expression of theinstant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translationinitiation codon and about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites NcoI (which includes the ATGtranslation initiation codon), SmaI, KpnI and XbaI. The entire cassetteis flanked by HindIII sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Assays for fructosyltransferase activity may be conducted under wellknown experimental conditions which permit optimal enzymatic activity.For example, assays for 1-FFT and 1-SST are presented by van der Meer etal. (1998) Plant J. 15:489-500. Assays for 6-SFT are presented bySprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92:11652-11656.

Example 8 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand Hind III sites was inserted at the BamHI site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the NdeI site at the position oftranslation initiation was converted to an NcoI site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10□g/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 250.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Assays for fructosyltransferase activity may be conducted under wellknown experimental conditions which permit optimal enzymatic activity.For example, assays for 1-FFT and 1-SST are presented by van der Meer etal. (1998) Plant J. 15:489-500. Assays for 6-SFT are presented bySprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92:11652-11656.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having 1-SST activity, wherein the amino acidsequence of the polypeptide and the amino acid sequence of SEQ ID NO:16have at least 98% sequence identity based on the Clustal alignmentmethod with default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5, or (b) a complement of the nucleotide sequence of(a), wherein the complement and the nucleotide sequence contain the samenumber of nucleotides and are 100% complementary.
 2. The polynucleotideof claim 1 wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:16.
 3. The polynucleotide of claim 1 wherein the nucleotidesequence comprises the nucleotide sequence of SEQ ID NO:15.
 4. A vectorcomprising the polynucleotide of claim
 1. 5. A recombinant DNA constructcomprising the polynucleotide of claim 1 operably linked to at least oneregulatory sequence.
 6. A method for transforming a cell comprisingtransforming a cell with the polynucleotide of claim
 1. 7. A cellcomprising the recombinant DNA construct of claim
 5. 8. A method forproducing a transgenic plant comprising transforming a plant cell withthe polynucleotide of claim 1 and regenerating a transgenic plant fromthe transformed plant cell.
 9. A plant comprising the recombinant DNAconstruct of claim
 5. 10. A seed comprising the recombinant DNAconstruct of claim
 5. 11. The polynucleotide of claim 1, wherein theamino acid sequence of the polypeptide has at least 99% sequenceidentity, based on the Clustal V method of alignment with the defaultparameters, when compared to SEQ ID NO:16.